Evolution of the 3-Hydroxypropionate Bicycle and Recent Transfer of Anoxygenic Photosynthesis Into the Chloroflexi

Evolution of the 3-hydroxypropionate bicycle and recent transfer of anoxygenic photosynthesis into the Chloroflexi Patrick M. Shiha,b,1, Lewis M. Wardc, and Woodward W. Fischerc,1 aFeedstocks Division, Joint BioEnergy Institute, Emeryville, CA 94608; bEnvironmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and cDivision of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125 Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved August 21, 2017 (received for review June 14, 2017) Various lines of evidence from both comparative biology and the provide a hard geological constraint on these analyses, the timing geologic record make it clear that the biochemical machinery for of these evolutionary events remains relative, thus highlighting anoxygenic photosynthesis was present on early Earth and provided the uncertainty in our understanding of when and how anoxy- the evolutionary stock from which oxygenic photosynthesis evolved genic photosynthesis may have originated. ca. 2.3 billion years ago. However, the taxonomic identity of these A less recognized alternative is that anoxygenic photosynthesis early anoxygenic phototrophs is uncertain, including whether or not might have been acquired in modern bacterial clades relatively they remain extant. Several phototrophic bacterial clades are thought recently. This possibility is supported by the observation that to have evolved before oxygenic photosynthesis emerged, including anoxygenic photosynthesis often sits within a derived position in the Chloroflexi, a phylum common across a wide range of modern the phyla in which it is found (3). Moreover, it is increasingly environments. Although Chloroflexi have traditionally been thought being recognized that horizontal gene transfer (HGT) has likely to be an ancient phototrophic lineage, genomics has revealed a much played a major role in the distribution of phototrophy (8–10). greater metabolic diversity than previously appreciated. Here, using a Together, these lines of evidence suggest that phototrophy may combination of comparative genomics and molecular clock analyses, have first evolved in lineages that are either yet to be discovered we show that phototrophic members of the Chloroflexi phylum are or have gone extinct. Similarly, although a number of modern- MICROBIOLOGY not particularly ancient, having evolved well after the rise of oxygen day phototrophic members of the Chloroflexi have been identi- (ca. 867 million years ago), and thus cannot be progenitors of oxy- fied, we cannot definitively draw the inference that their ancestors genic photosynthesis. Similarly, results show that the carbon fixation must also have had photosynthetic metabolisms. This reasoning pathway that defines this clade—the 3-hydroxypropionate bicycle— highlights the nuances and challenges in interpreting the traits of evolved late in Earth history as a result of a series of horizontal gene ancient organisms, and the uncertainty inherent in attributing the transfer events, explaining the lack of geological evidence for this evolution of photosynthesis to bacterial lineages that may have pathway based on the carbon isotope record. These results demon- existed billions of years ago. strate the role of horizontal gene transfer in the recent metabolic Reconstructing evolutionary histories over large geological innovations expressed within this phylum, including its importance EARTH, ATMOSPHERIC, timescales can be complicated, with issues stemming from HGT, AND PLANETARY SCIENCES in the development of a novel carbon fixation pathway. deep coalescence, gene duplication, and extinction (11). Thus, older events are inherently more difficult to study. These hurdles carbon fixation | phototrophy | molecular clock | comparative genomics make it challenging to piece together not only the evolution of phototrophy but also the origins of the six known carbon fixation rom both biological and geological data, it is widely thought pathways (12, 13), as they may have been some of the earliest Fthat anoxygenic photosynthesis preceded the development of metabolisms to evolve (14–18). To this end, the antiquity of these oxygenic photosynthesis and the rise of atmospheric oxygen (1). metabolisms has hampered efforts to retrace their origins and Although it has been largely accepted that oxygenic photosyn- identify what pathway was used by the earliest photoautotrophs. For thesis evolved in ancient Cyanobacterial lineages (2), very little is known about the nature and evolutionary history of anoxygenic Significance phototrophy, with most of our understanding stemming from assumptions and hypotheses based on the few extant bacterial Photosynthesis supports life on our planet; however, we know taxa that host this metabolism. Given the significance of the ca. ∼ very little about the origins of this metabolism. Anoxygenic accumulation of oxygen 2.3 billion years ago, it is of no photosynthesis existed prior to the evolution of its more complex surprise that the majority of efforts have focused on studying counterpart, oxygenic photosynthesis. It is not known which oxygenic photosynthesis. However, this has resulted in a paucity groups of microbes performed anoxygenic photosynthesis on of studies critically examining basic and core questions on the early Earth; one idea has argued that anoxygenic photosynthesis origins of anoxygenic photosynthesis, such as what bacterial evolved in the bacterial phylum Chloroflexi. We compared the lineage developed this metabolism and when did it evolve? genomes of different members of the Chloroflexi, finding that These questions are essential to our first-order knowledge of acquisition of photosynthesis and their unique carbon fixation how early microbial metabolisms may have shaped the geo- metabolism evolved remarkably late in Earth history—atime chemical cycles of our planet. long after the rise of oxygen in the atmosphere. Although chlorophyll-based photosynthesis can be found in seven known bacterial phyla (i.e., Cyanobacteria, Proteobacteria, Author contributions: P.M.S., L.M.W., and W.W.F. designed research, performed research, Chlorobi, Firmicutes, Acidobacteria, Gemmatimonadetes, and analyzed data, and wrote the paper. Chloroflexi) (3), a number of studies have argued that one of the The authors declare no conflict of interest. earliest forms of anoxygenic photosynthesis arose within the This article is a PNAS Direct Submission. Chloroflexi phylum before the invention of oxygenic photosyn- 1To whom correspondence may be addressed. Email: [email protected] or wfischer@ thesis during the Archean Eon (4–7). These analyses are based caltech.edu. on phylogenies generated from genes involved in photosynthesis. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. However, without the inclusion of valuable dating information to 1073/pnas.1710798114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1710798114 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 example, the widespread diversity of bacteria containing the enzyme Results and Discussion ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) (19) Cross-Calibrated Molecular Clock Analyses on the Chloroflexi Phylum. makes it difficult to definitively determine which clade first evolved Phylogenies quantify the evolutionary relationships between this enzyme and the Calvin−Benson cycle. different groups; however, these approaches typically provide The phylum Chloroflexi presents a unique opportunity to in- little information on the absolute timing of evolutionary diver- vestigate a number of hypotheses central to our understanding of gences. Molecular clocks provide a means to include time con- the early evolution of photosynthesis and carbon fixation. The straints into these analyses to test hypotheses for both the evolution first described Chloroflexi were discovered and characterized for and timing of specific events. We have previously demonstrated their ability to perform both anoxygenic photosynthesis and a lower uncertainties and increased accuracy in dating deep evolu- unique carbon fixation metabolism—the 3-hydroxypropionate tionary events using cross-calibrated molecular clock analyses (25), (3HP) bicycle (20–22). Thus, these two metabolic traits have long specifically by tying bacterial (Proteobacteria and Cyanobacteria) been assumed to be tied to the origins of the evolution of the divergences to the eukaryotic plant and algal fossil records (via Chloroflexi phylum (4, 7). Importantly, a key piece of evolu- mitochrondria and plastids) and leveraging these calibrations across tionary insight can be obtained by better understanding the the tree. While estimated uncertainties for deep divergences remain timing of these events in a geological context. Estimating the age largeincomparisonwithgeochronological constraints, this ap- and origin of photoautotrophic clades within the Chloroflexi proach enables us to test hypotheses that place the evolutionary phylum may provide key constraints to help resolve when pho- timing of phototrophy and the 3HP bicycle in the Chloroflexi be- tosynthesis and the 3HP bicycle evolved. However, this task is fore the rise of oxygen versus those that would place these evolu- challenging, because, unlike other phyla, Chloroflexi lack specific tionary events after it. diagnostic molecular biomarkers or microfossil morphologies to To construct the molecular dataset, an alignment

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